Transamidations are calcium-dependent reactions catalyzed by transglutaminase enzymes. Transglutaminases utilize the 3-carboxyamide group of peptide-bound glutamine residues as acyl donor, having a broad specificity for acceptor substrates. Extensive research has investigated the role of TGC in the fatal neurodegenerative diseases (e.g. Alzheimer's disease) and cancer. The molecular mechanism by which TGC contributes to the etiology of these diseases has not been discovered. It may be that a small fraction of transglutaminase is active in a disease state, and the lack of data correlate transglutaminase to its biological function makes it difficult or not possible to predict the enzyme molecular mechanism. The transamidation activity of TGC was found to be located in the N terminal sequences.TGC (80 kDa) is inactive in cross-linking and converted in a small fraction in vitro and in vivo to a cross-linking active isoform (TG), with a molecular weight of about 55 kDa. It has been assumed that oligomer formation by transglutaminase is toxic and involved in cell death, although there are few experimental data that directly test this hypothesis. To test this hypothesis, the principal investigator proposes the following aims 1) Induction of protein cross-linking by the active TG that has never been used in cellular transfection, 2) Identification of cross-linked mitochondrial protein(s) from the induced cells 3) Isolation and purification of cross-linked proteins from cells with high apoptosis indices. The experimental approach for testing the role of tansamidation involvement in the early stages of cell death will be: Eukaryotic expression vectors containing TG cDNA will be used to transiently transfect human breast cancer MCF-7, T47D cells which express low amounts of transglutaminase. The transamidation effect on apoptosis will be examined by using Fluorescence-Activated Cell Sorting (FACS). Transamidated proteins will be purified by affinity columns and identified by immuno-blotting and sequencing. The effect of increasing calcium levels on induction of apoptosis independently of transamidation will be tested by a) specific TG inhibitors (polyamines, such as cystamine), b) antisense transfection and siRNA to block transglutaminase expression, and c) Site-directed mutagenesis of the isoform cDNAs will be used to eliminate transamidation active site and the mutated cDNA expression vectors and active isoforms cDNAs will be used to transfect cells separately.